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Results for “Multiprotein Complexes”

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PubMed2003

Heat-shock proteins as regulators of apoptosis.

Heat-shock proteins are produced in response to different types of stress conditions making cells resistant to stress-induced cell damage. Under normal conditions, heat-shock proteins play numerous roles in cell function, including modulating protein activity by changing protein conformation, promoting multiprotein complex assembly/disassembly, regulating protein degradation within the proteasome pathway, facilitating protein translocation across organellar membranes, and ensuring proper folding of nascent polypeptide chains during protein translation. When cells are stressed, a common response is to undergo cell death by one of two pathways, either 'necrosis' or 'apoptosis'. Recently, both routes to cell death have been revealed to share similar mechanisms, with heat-shock proteins and their cofactors responsible for inhibiting both apoptotic and necrotic pathways. We therefore briefly summarize recent reports showing molecular evidence of cell death regulation by heat-shock proteins and their cochaperones.

Animals↗

PubMed2004

Connexin43 pseudogene is expressed in tumor cells and inhibits growth.

Pseudogenes are classically thought of as nonfunctional DNA sequences due to their inability to be translated, or to produce a functional protein. Gap junctions, a multiprotein complex made of proteins called connexins, are involved in intercellular communication and are deregulated in many cancers. Connexin43 (Cx43) is the only connexin for which a pseudogene has been reported so far. The Cx43 pseudogene (PsiCx43) has all of the features of an expressed gene. We identified the presence of a PsiCx43 mRNA transcript in several cancer cell lines and in none of the normal mammary epithelial cells studied. Using an in vitro translation assay, we found that the PsiCx43 coding plasmid could be translated into a 43 kDa protein. This was further confirmed by expressing a PsiCx43-green fluorescence protein fusion protein in breast cancer MCF-7 cells. We then examined the functional significance of the PsiCx43. In both MTT growth and colony formation assays, significant growth inhibition was observed, a feature common to cells overexpressing the Cx43 gene. However, using a scrape-loading assay, we could not detect any effect on gap junctional intercellular communication. Based on our findings, PsiCx43 joins and enlarges the thus far restricted group of functionally transcribed and translated pseudogenes.

Amino Acid Sequence↗

PubMed2004

WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy.

WD-repeat proteins are regulatory beta-propeller platforms that enable the assembly of multiprotein complexes. Here, we report the functional and bioinformatic analysis of human WD-repeat protein Interacting with PhosphoInosides (WIPI)-1alpha (WIPI49/Atg18), a member of a novel WD-repeat protein family with autophagic capacity in Saccharomyces cerevisiae and Caenorhabditis elegans, recently identified as phospholipid-binding effectors. Our phylogenetic analysis divides the WIPI protein family into two paralogous groups that fold into 7-bladed beta-propellers. Structural modeling identified two evolutionary conserved interaction sites in WIPI propellers, one of which may bind phospholipids. Human WIPI-1alpha has LXXLL signature motifs for nuclear receptor interactions and binds androgen and estrogen receptors in vitro. Strikingly, human WIPI genes were found aberrantly expressed in a variety of matched tumor tissues including kidney, pancreatic and skin cancer. We found that endogenous hWIPI-1 protein colocalizes in part with the autophagosomal marker LC3 at punctate cytoplasmic structures in human melanoma cells. In addition, hWIPI-1 accumulated in large vesicular and cup-shaped structures in the cytoplasm when autophagy was induced by amino-acid deprivation. These cytoplasmic formations were blocked by wortmannin, a classic inhibitor of PI-3 kinase-mediated autophagy. Our data suggest that WIPI proteins share an evolutionary conserved function in autophagy and that autophagic capacity may be compromised in human cancers.

Amino Acid Sequence↗

PubMed2005

Poly(ADP-ribose): a co-regulator of DNA methylation?

The formation of multiprotein complexes is l'ordre du jour in regulatory pathways. In this issue of Oncogene, Reale et al. report the formation of a particularly sophisticated complex of two important regulatory enzymes, DNMT1 (DNA methyltransferase-1) and PARP-1 (poly(ADP-ribose)polymerase-1). The former evolved with a specific sequence motif binding the enzymatic product of the latter. The product, poly(ADP-ribose), bonds the two partners into a heterodimeric complex and, as a consequence, the catalytic function of DNMT1 is silenced. Thus, PARP-1 becomes a conditional negative regulator of DNMT1. In a larger perspective, Reale et al. highlight the potential role of PARP-1 as a co-regulator of DNA methylation leading to epigenetic reprogramming of cancer cells.

Animals↗

PubMed2005

The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin.

Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid studies, ENL interacted with AF4 and AF5q31 as well as with a fragment of AF10. A structure-function analysis revealed that the AF4/AF5q31/AF10 binding domain in ENL coincided with the C-terminus that is essential for transformation by MLL-ENL. The ENL/AF4 association was corroborated by GST-pulldown experiments and by mutual coprecipitation. Both proteins colocalized in vivo in a nuclear speckled pattern. Moreover, AF4 and ENL coeluted on sizing columns together with the known ENL binding partner Polycomb3, suggesting the presence of a multiprotein complex. The overexpression of ENL alone activated a reporter construct and a mutational screen indicated the conserved YEATS domain as essential for this function. Overlay and pulldown-assays finally showed a specific and YEATS domain-dependent association of ENL with histones H3 and H1. In summary, our studies support a common role for nuclear MLL fusion partners in chromatin biology.

Animals↗

PubMed2006

Insulin and amino-acid regulation of mTOR signaling and kinase activity through the Rheb GTPase.

Target of Rapamycin (TOR), a giant protein kinase expressed by all eucaryotic cells, controls cell size in response to nutrient signals. In metazoans, cell and organismal growth is controlled by nutrients and the insulin/insulin-like growth factor (IGF) system, and the understanding of how these inputs coordinately regulate TOR signaling has advanced greatly in the past 5 years. In single-cell eucaryotes and Caenorhabditis elegans, TOR is a dominant regulator of overall mRNA translation, whereas in higher metazoans, TOR controls the expression of a smaller fraction of mRNAs that is especially important to cell growth. TOR signals through two physically distinct multiprotein complexes, and the control of cell growth is mediated primarily by TOR complex 1 (TORC1), which contains the polypeptides raptor and LST8. Raptor is the substrate binding element of TORC1, and the ability of raptor to properly present substrates, such as the translational regulators 4E-BP and p70 S6 kinase, to the TOR catalytic domain is essential for their TOR-catalysed phosphorylation, and is inhibited by the Rapamycin/FKBP-12 complex. The dominant proximal regulator of TORC1 signaling and kinase activity is the ras-like small GTPase Rheb. Rheb binds directly to the mTOR catalytic domain, and Rheb-GTP enables TORC1 to attain an active configuration. Insulin/IGF enhances Rheb GTP charging through the ability of activated Akt to inhibit the Rheb-GTPase-activating function of the tuberous sclerosis heterodimer (TSC1/TSC2). Conversely, energy depletion reduces Rheb-GTP charging through the ability of the adenosine monophosphate-activated protein kinase to phosphorylate TSC2 and stimulate its Rheb-GTPase activating function, as well as by HIFalpha-mediated transcriptional responses that act upstream of the TSC1/2 complex. Amino-acid depletion inhibits TORC1 acting predominantly downstream of the TSC complex, by interfering with the ability of Rheb to bind to mTOR. The components of the insulin/IGF pathway to TORC1 are now well established, whereas the elements mediating the more ancient and functionally dominant input of amino acids remain largely unknown.

Animals↗

PubMed2001

GSK3 takes centre stage more than 20 years after its discovery.

Identified originally as a regulator of glycogen metabolism, glycogen synthase kinase-3 (GSK3) is now a well-established component of the Wnt signalling pathway, which is essential for setting up the entire body pattern during embryonic development. It may also play important roles in protein synthesis, cell proliferation, cell differentiation, microtubule dynamics and cell motility by phosphorylating initiation factors, components of the cell-division cycle, transcription factors and proteins involved in microtubule function and cell adhesion. Generation of the mouse knockout of GSK3beta, as well as studies in neurons, also suggest an important role in apoptosis. The substrate specificity of GSK3 is unusual in that efficient phosphorylation of many of its substrates requires the presence of another phosphorylated residue optimally located four amino acids C-terminal to the site of GSK3 phosphorylation. Recent experiments, including the elucidation of its three-dimensional structure, have enhanced our understanding of the molecular basis for the unique substrate specificity of GSK3. Insulin and growth factors inhibit GSK3 by triggering its phosphorylation, turning the N-terminus into a pseudosubstrate inhibitor that competes for binding with the 'priming phosphate' of substrates. In contrast, Wnt proteins inhibit GSK3 in a completely different way, by disrupting a multiprotein complex comprising GSK3 and its substrates in the Wnt signalling pathway, which do not appear to require a 'priming phosphate'. These latest findings have generated an enormous amount of interest in the development of drugs that inhibit GSK3 and which may have therapeutic potential for the treatment of diabetes, stroke and Alzheimer's disease.

Animals↗

PubMed2002

A protein-domain microarray identifies novel protein-protein interactions.

Protein domains mediate protein-protein interactions through binding to short peptide motifs in their corresponding ligands. These peptide recognition modules are critical for the assembly of multiprotein complexes. We have arrayed glutathione S-transferase (GST) fusion proteins, with a focus on protein interaction domains, on to nitrocellulose-coated glass slides to generate a protein-domain chip. Arrayed protein-interacting modules included WW (a domain with two conserved tryptophans), SH3 (Src homology 3), SH2, 14.3.3, FHA (forkhead-associated), PDZ (a domain originally identified in PSD-95, DLG and ZO-1 proteins), PH (pleckstrin homology) and FF (a domain with two conserved phenylalanines) domains. Here we demonstrate, using peptides, that the arrayed domains retain their binding integrity. Furthermore, we show that the protein-domain chip can 'fish' proteins out of a total cell lysate; these domain-bound proteins can then be detected on the chip with a specific antibody, thus producing an interaction map for a cellular protein of interest. Using this approach we have confirmed the domain-binding profile of the signalling molecule Sam68 (Src-associated during mitosis 68), and have identified a new binding profile for the core small nuclear ribonucleoprotein SmB'. This protein-domain chip not only identifies potential binding partners for proteins, but also promises to recognize qualitative differences in protein ligands (caused by post-translational modification), thus getting at the heart of signal transduction pathways.

Amino Acid Sequence↗

PubMed2003

Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.

The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.

Allosteric Regulation↗

PubMed2005

Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells.

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein-protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated 'signalling particles' with an estimated size of 60-75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.

Alternative Splicing↗

PubMed2006

Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death.

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.

Animals↗

PubMed2007

The plastome-encoded PsaJ subunit is required for efficient Photosystem I excitation, but not for plastocyanin oxidation in tobacco.

The functions of several small subunits of the large photosynthetic multiprotein complex PSI (Photosystem I) are not yet understood. To elucidate the function of the small plastome-encoded PsaJ subunit, we have produced knockout mutants by chloroplast transformation in tobacco (Nicotiana tabacum). PsaJ binds two chlorophyll-a molecules and is localized at the periphery of PSI, close to both the Lhca2- and Lhca3-docking sites and the plastocyanin-binding site. Tobacco psaJ-knockout lines do not display a visible phenotype. Despite a 25% reduction in the content of redox-active PSI, neither growth rate nor assimilation capacity are altered in the mutants. In vivo, redox equilibration of plastocyanin and PSI is as efficient as in the wild-type, indicating that PsaJ is not required for fast plastocyanin oxidation. However, PsaJ is involved in PSI excitation: altered 77 K chlorophyll-a fluorescence emission spectra and reduced accumulation of Lhca3 indicate that antenna binding and exciton transfer to the PSI reaction centre are impaired in DeltapsaJ mutants. Under limiting light intensities, growth of DeltapsaJ plants is retarded and the electron-transport chain is far more reduced than in the wild-type, indicating that PSI excitation might limit electron flux at sub-saturating light intensities. In addition to defining in vivo functions of PsaJ, our data may also have implications for the interpretation of the crystal structure of PSI.

Kinetics↗

PubMed1998

Demonstration of a RNA-dependent nuclear interaction between the promyelocytic leukaemia protein and glyceraldehyde-3-phosphate dehydrogenase.

The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH-PML interaction may be involved in the regulation of apoptosis.

3T3 Cells↗

PubMed1999

Mitochondrial control of apoptosis: an overview.

The mitochondrial permeability transition (PT) pore, also called the mitochondrial megachannel, is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes, exactly the same location at which Bax, Bcl-2 and Bcl-XL are particularly abundant. The PT pore participates in the regulation of matrix Ca2+, pH, transmembrane potential and volume, and functions as a Ca(2+)-, voltage-, pH- and redox-gated channel with several levels of conductance and little, if any, ion selectivity. We have obtained three independent lines of evidence implicating the mitochondrial PT pore in apoptosis. First, in intact cells, apoptosis is accompanied by an early dissipation of the mitochondrial transmembrane potential, delta psi m. In several models of apoptosis, specific agents inhibiting the mitochondrial PT pore abolish this dissipation of the delta psi m and simultaneously prevent activation of downstream caspases and endonucleases, indicating that PT pore opening can be a critical event of the apoptotic process. Secondly, mitochondria are rate-limiting for caspase and nuclease activation in several cell-free systems of apoptosis. Isolated mitochondria release apoptogenic factors capable of activating pro-caspases or endonucleases upon opening of the mitochondrial megachannel in vitro. Thirdly, opening of the purified PT pore complex reconstituted into liposomes is inhibited by recombinant Bcl-2 or Bcl-XL, two apoptosis-inhibitory proteins that also prevent PT pore opening in cells and isolated mitochondria. Altogether, our results suggest that PT pore opening is sufficient and (mostly) necessary for triggering apoptosis. The implications of these findings are examined in the light of pharmacological interventions in apoptosis.

Animals↗

PubMed2003

Signalling networks, inflammation and innate immunity.

We have been analysing the signalling systems that couple to receptors of the TIR (Toll/interleukin-1 receptor) family, which signal through a common cytoplasm region; the TIR domain. These systems are of both practical and fundamental biological significance, being central to the pathogenesis of chronic inflammatory diseases such as atherosclerosis, to host defence throughout the biological world, and are ancient in the context of life on earth, having originated more than 1 billion years ago: prior to the divergence of plants and animals. TIR domain receptors couple to at least two sets of well-characterized pathways: those leading to the activation of inhibitory kappaB kinase complexes/nuclear factor kappaB, and those leading to the activation of mitogen-activated protein kinase/AP-1/ATF-2 etc. We have been investigating these systems using a combination of expression screening methods to identify new components, and real-time green fluorescent protein-based techniques to observe execution of signalling programmes in real time. Our data reveal that there is a very large level of cell-to-cell variation in signal programme execution even in clonal populations and that at least one mechanism for dealing with this heterogeneity is the assembly of signal transduction components into large multiprotein complexes.

Humans↗

PubMed2005

Spatiotemporal regulation of the Pak1 kinase.

Pak1 (p21-activated kinase 1) is a key regulator of the actin cytoskeleton, adhesion and cell motility. Such biological roles require a tight spatial and kinetic control of its localization and activity. We summarize here the current knowledge on Pak1 dynamics in vivo. Inactive dimeric Pak1 is mainly cytosolic. Localized interaction with the activators Cdc42-GTP and Rac1-GTP stimulates the kinase at the sites of cellular protrusions. Moreover, Pak1 is dynamically engaged into multiprotein complexes forming adhesions to the extracellular matrix. Cutting edge microscopy technologies on living cells are finally shedding light on the intricate spatiotemporal mechanisms regulating Pak1.

Animals↗

PubMed2006

Peptides for disruption of PKA anchoring.

Adaptor or scaffolding proteins are at the basis of multiprotein complexes that spatially and temporally co-ordinate the propagation and integration of a broad range of cellular events. One class of scaffolding proteins are AKAPs (A-kinase-anchoring proteins). They sequester PKA (protein kinase A) and other signalling molecules including phosphodiesterases, other protein kinases and protein phosphatases to specific subcellular compartments. AKAP-dependent protein-protein interactions play a role in many physiologically relevant processes. For example, AKAP-PKA interactions are essential for the vasopressin-mediated water re-absorption in renal collecting duct principal cells or beta-adrenoceptor-induced increases in cardiac myocyte contractility. Here, we discuss recently developed peptide disruptors of AKAP-PKA interactions. Such peptides are valuable tools to study the relevance of PKA anchoring in cellular processes.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Regulation of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) catalyses the formation of malonyl-CoA, an essential substrate for fatty acid synthesis in lipogenic tissues and a key regulatory molecule in muscle, brain and other tissues. ACC contributes importantly to the overall control of energy metabolism and has provided an important model to explore mechanisms of enzyme control and hormone action. Mammalian ACCs are multifunctional dimeric proteins (530-560 kDa) with the potential to further polymerize and engage in multiprotein complexes. The enzymatic properties of ACC are complex, especially considering the two active sites, essential catalytic biotin, the three-substrate reaction and effects of allosteric ligands. The expression of the two major isoforms and splice variants of mammalian ACC is tissue-specific and responsive to hormones and nutritional status. Key regulatory elements and cognate transcription factors are still being defined. ACC specific activity is also rapidly modulated, being increased in response to insulin and decreased following exposure of cells to catabolic hormones or environmental stress. The acute control of ACC activity is the product of integrated changes in substrate supply, allosteric ligands, the phosphorylation of multiple serine residues and interactions with other proteins. This review traces the path and implications of studies initiated with Dick Denton in Bristol in the late 1970s, through to current proteomic and other approaches that have been consistently challenging and immensely rewarding.

Acetyl-CoA Carboxylase↗